Cell culture microcarriers

ABSTRACT

Improved cell culture microcarriers, and methods for their production and use, are disclosed herein. These improved microcarriers have positive charge capacities adjusted and/or controlled within a range suitable for good cell growth. One method for producing such improved microcarriers is by treating beads formed from polymers containing pendant hydroxy groups, such as dextran beads, with an aqueous solution of an alkaline material and a chloro- or bromo-substituted tertiary amine under precisely controlled conditions to produce the desired exchange capacity. The resultant positively charged microcarriers have been used in microcarrier cultures to produce outstanding growth of anchorage-dependent cells. Such cells can be harvested, or used for the production of viruses, vaccines, hormones, interferon or other cellular growth by-products.

GOVERNMENT SUPPORT

The Government has rights in this invention pursuant to NSF Grant No.BMS 7405676A01 and NIEHS Grant No. TO1ES 00063.

RELATED APPLICATION

This is a division, of application Ser. No. 842,696, filed Oct. 17,1977, now U.S. Pat. No. 4,189,534 which in turn is acontinuation-in-part of Ser. No. 740,993, filed Nov. 11, 1976, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of biology and more particularly in thefield of cell biology.

2. Description of the Prior Art

The ability to grow mammalian cells is important at both the laboratoryand industrial levels. At the laboratory level, the limiting factor forcellular or viral research at the sub-cellular level is often the amountof raw material available to be studied. At the industrial level, thereis much effort being devoted to the development of pharmaceuticals basedon mammalian cell products. These are primarily vaccines for human oranimal viruses, but also include human growth hormone and other bodyhormones and biochemicals for medical applications.

Some mammalian cell types have been adapted for growth in suspensioncultures. Examples of such cell types include HeLa (human), BHK (babyhamster kidney) and L cells (mouse). Such cells, in general, havenon-normal genetic complements, i.e., too many or too few chromosomes orabnormal chromosomes. Often, these cells will produce a tumor uponinjection into an animal of the appropriate species.

Other mammalian cell types have not been adapted for growth insuspension culture to date, and will grow only if they can becomeattached to an appropriate surface. Such cell types are generally termed"anchorage-dependent" and include 3T3 mouse fibroblasts, mouse bonemarrow epithelial cells; Murine leukemia virus-producing strains ofmouse fibroblasts, primary and secondary chick fibroblasts; WI-38 humanfibroblast cells; and, normal human embryo lung fibroblast cells(HEL299, ATCC #CCL137). Some anchorage-dependent cells have been grownwhich are tumor causing but others were grown and found to be non-tumorcausing. Also, some anchorage-dependent cells, such as WI-38 and HEL299,can be grown which are genetically normal.

Whereas considerable progress has been made in large scale mammaliancell propagation using cell lines capable of growth in suspensionculture, progress has been very limited for large scale propagation ofanchorage-dependent mammalian cells. Previous operational techniquesemployed for large scale propagation of anchorage-dependent cells werebased on linear expansion of small scale processes. Cell culture plantsutilized a large number of low yield batch reactors, in the forms ofdishes, prescription bottles, roller tubes and roller bottles. Each ofthese was a discrete unit or isolated batch reactor requiring individualenvironmental controls. These controls, however, were of the mostprimitive type due to economic considerations. Variation in nutrientswas corrected by a medium change, an operation requiring two steps,i.e., medium removal and medium addition. Since it was not uncommon fora moderately sized facility to operate hundreds of these batch reactorsat a time, even a single change of medium required hundreds ofoperations, all of which had to be performed accurately, and underexacting sterile conditions. Any multiple step operation, such as celltransfer or harvest, compounded the problem accordingly. Thus, costs ofequipment, space and manpower were great for this type of facility.

There are alternative methods to linear scale-up from small batchcultures which have been proposed. Among such alternatives which havebeen reported in the literature are plastic bags, stacked plates, spiralfilms, glass bead propagators, artificial capillaries, andmicrocarriers. Among these, microcarrier systems offer certainoutstanding and unique advantages. For example, great increases in theattainable ratio of growth surface to vessel volume (S/V) can beobtained using microcarriers over both traditional and newly developedalternative techniques. The increase in S/V attainable allows theconstruction of a single-unit homogeneous or quasi-homogeneous batch orsemi-batch propagator for high volumetric productivity. Thus, a singlestirred tank vessel with simple feedback control for pH and pO₂ presentsa homogeneous environment for a large number of cells therebyeliminating the necessity for expensive and space consuming, controlledenvironment incubators. Also, the total number of operations requiredper unit of cells produced is drastically reduced. In summary,microcarriers seem to offer economies of capital, space and manpower inthe production of anchorage-dependent cells, relative to currentproduction methods.

Microcarriers also offer the advantage of environmental continuity sincethe cells are grown in one controlled environment. Thus, microcarriersprovide the potential for growing anchorage-dependent mammalian cellsunder one set of environmental conditions which can be regulated toprovide constant, optimal cell growth.

One of the more promising microcarrier systems to date has been reportedby van Wezel and involves the use of diethylaminoethyl(DEAE)-substituted dextran beads in a stirred tank. A. L. van Wezel,"Growth of Cell Strains and Primary Cells on Microcarriers inHomogeneous Culture," Nature 216:64 (1967); D. van Hemert, D. G. Kilburnand A. L. van Wezel, "Homogeneous Cultivation of Animal Cells for theProduction of Virus and Virus Products," Biotechnol. Bioeng. 11:875(1969); and A. L. van Wezel, "Microcarrier Cultures of Animal Cells,"Tissue Culture, Methods and Applications, P. F. Kruse and M. K.Patterson, eds., Academic Press, New York, p. 372 (1973). These beadsare commercially produced by Pharmacia Fine Chemicals, Inc., Piscataway,N.J., under the tradename DEAE-Sephadex A50, an ion exchange system.Chemically, these beads are formed from a crosslinked dextran matrixhaving diethylaminoethyl groups covalently bound to the dextran chains.As commercially available, DEAE-Sephadex A50 beads are believed to havea particle size of 40-120 μm and a positive charge capacity of about 5.4meq per gram of dry, crosslinked dextran (ignores weight of attachedDEAE moieties). Other anion exchange resins, such as DEAE-Sephadex A25,QAE-Sephadex A50 and QAE-Sephadex A25 were also stated by van Wezel tosupport cell growth.

The system proposed by van Wezel combines multiple surfaces with movablesurfaces and has the potential for innovative cellular manipulations andoffers advantages in scale-up and environmental controls. Despite thispotential, these suggested techniques have not been significantlyexploited because researchers have encountered difficulties in cellproduction due to certain deleterious effects caused by the beads. Amongthese are initial cell death among a high percentage of the cellinoculum and inadequate cell growth even for those cells which attach.The reasons for these deleterious effects are not thoroughly understood,although it has been proposed that they may be due to bead toxicity ornutrient adsorption. See van Wezel, A. L. (1967), Nature 216: 64-65; vanWezel, A. L. (1973), Tissue Culture, Methods and Applications. Kruse, P.R. and Patterson, M. R. (eds.), pp. 372-377, Academic Press, New York;van Hemert, P., Kilburn, D. G., and van Wezel, A. L. (1969), Biotechnol.Bioeng. 11: 875-885; Horng, C. and McLimans, W. (1975), Biotechnol.Bioeng. 17: 713-732.

It could be that the deleterious effects of these commercially availableion exchange resins are due to their method of manufacture. Certain ofthese production methods are described for polyhydroxy materials inpatents such as: U.S. Pat. Nos. 3,277,025; 3,275,576; 3,042,667 and3,208,994 all to Flodin et al. Whatever the reason, however, thepresently commercially available materials are simply not sufficient forgood cell growth of a wide variety of cell types.

One solution to overcoming some of the deleterious effects encounteredin attempts to use such commercially available microcarriers for cellgrowth is described in U.S. Pat. No. 4,036,693, issued on July 19, 1977to Levine et al. Therein, a method for treating these commerciallyavailable ion exchange resins with macromolecular polyanions, such ascarboxymethylcellulose, is proposed. While this method has provensuccessful, it would clearly be more advantageous if the beads could bemanufactured initially to have properties designed for outstandinggrowth of anchorage-dependent cells.

SUMMARY OF THE INVENTION

It has now been discovered that the charge capacity of microcarriers hasto be adjusted and/or controlled within a certain range to result ingood growth of a wide variety of anchorage-dependent cell types atreasonable microcarrier concentrations. Based upon this discovery,microcarrier beads have been produced with controlled charge capacitiesand such beads have been used to obtain good growth of a variety ofanchorage-dependent cells. Cells grown using such microcarrier systemscan be harvested or used in the production of animal or plant viruses,vaccines, hormones, interferon or other cell growth by-products.

One example of the improved microcarriers is those produced usingpolymers with pendant hydroxy groups, such as crosslinked dextran beads.These beads can be treated with an aqueous solution of a tertiary orquaternary amine, such as diethylaminoethylchloride:chloride, and analkaline material, such as sodium hydroxide. The specific chargecapacity of the beads is controlled by varying the absolute amounts ofthe dextran, tertiary amine salt and alkalne material, the ratio ofthese materials, and/or the time and temperature of treatment.

Microcarriers produced according to this invention can be used incultures without the high initial cell loss heretofore experienced withcommercially available microcarriers. Additionally, attached cellsspread and grow to confluence on the beads reaching extremely high cellconcentrations in the suspending medium. The concentration ofmicrocarriers in suspension is not limited to very low levels as wascustomary with the prior art materials, and cell growth appears only tobe limited by factors which do not appear to be associated with themicrocarriers. Because of this, great increases in the volumetricproductivity of cell cultures can be obtained. In short, the potentialoffered from the use of microcarriers in the growth of cells, andparticularly anchorage-dependent cells, can now be realized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot graphically illustrating the growth characteristics ofnormal diploid human embryo lung fibroblast cells (HEL299) at amicrocarrier concentration of 2 grams dry, crosslinked dextran/liter forboth commercially available DEAE-treated dextran microcarriers andDEAE-treated microcarriers produced according to this invention;

FIG. 2 graphically illustrates the growth characteristics of both normaldiploid human embryo lung fibroblast cells (HEL299) and secondarychicken embryo fibroblasts at a microcarrier concentration of 5 gramsdry, crosslinked dextran/liter using improved DEAE-treated microcarriersof this invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, the terms "microcarriers," "cell-culture microcarriers"and "cell-growth microcarriers" mean small, discrete particles suitablefor cell attachment and growth. Often, although not always,microcarriers are porous beads which are formed from polymers. Usually,cells attach to and grow on the outer surfaces of such beads.

As previously described, it has now been discovered that the amount ofcharge capacity on cell culture microcarriers must be adjusted and/orcontrolled to be within a certain range for adequate cell growth atreasonable microcarrier concentrations. Suitable operating and preferredranges will vary with such factors as the specific cells to be grown,the nature of the microcarriers, the concentration of microcarriers, andother culture parameters including medium composition. In all cases,however, the amount of charge capacity which has been found to besuitable is significantly below the amounts believed to be present oncommercially available anion exchange resins previously suggested formicrocarrier cell cultures. For example, it is believed that theDEAE-Sephadex A50 beads, suggested by van Wezel, have a charge capacityof about 5.4 meq/gram of dry, untreated (without DEAE), crosslinkeddextran. In contradistinction to this relatively high charge capacity,microcarriers have been produced and found suitable for good cell growthaccording to this invention which have between about 0.1 and about 4.5meq/gram of dry, untreated microcarriers. Below about 0.1 meq/gram, itis believed that cells would have difficulty attaching to themicrocarriers. Above about 4.5 meq/gram, losses of initial cell inoculumtake place, and even the surviving cells do not grow well, particularlyat relatively high microcarrier concentrations.

For the growth of normal diploid human fibroblasts on crosslinkeddextran microcarriers, it has been found that a preferred range ofcharge capacity supplied by DEAE groups is from about 1.0 to about 2.8meq/gm of dry, untreated crosslinked dextran. While the preferred rangemay vary with different cell types or culture conditions, it is believedthat the preferred ranges for any given set of conditions will be withinthe 0.1-4.5 meq/gm range. The preferred and optimum conditions can bedetermined by a person skilled in the art for any set of conditions byroutine experimentation.

It will be recognized, of course, that there are certain deficiencies inattempting to define the charge capacity of microcarriers strictly on aunit weight basis. For example, two beads identical in every way exceptthat they are formed from materials having different densities with thesame charge distribution thereon would yield different values for theircharge capacity per unit weight. Similarly, two beads having identicalcharge capacities per unit weight might have quite different chargedistributions thereon.

An alternative definition can be made by specifying the range ofsuitable charges in terms of charge capacity per unit weight ofmicrocarriers in their final functional form. This basis would take intoaccount such factors as the weight of attached DEAE or other positivelycharged groups, as well as hydration of the beads, etc., whereas theprior definition is based on dry, crosslinked dextran and does not takesuch factors into account. In an aqueous cell culture medium, thedensity of microcarriers should be close to 0.1 gram/cc so that themicrocarriers can be readily dispersed throughout the culture. Basedupon this, it has been determined that the range of suitable chargecapacities for microcarriers of this invention defined in this way isfrom about 0.012 to about 0.25 meq/gram.

The ranges of suitable charge capacities previously specified on aweight basis are valid assuming the microcarriers have a substantiallyuniform charge distribution throughout their bulk. If the chargedistribution is uneven, it might be possible to have suitablemicrocarriers having charge capacities outside of those ranges. Theimportant criterion is, of course, that the charge capacity be adjustedto and/or controlled at a value sufficient to allow good cell growth onthe microcarriers.

Since it may be the charge pattern on the outer surface which isimportant, it is also desirable to be able to define a suitable chargecapacity range in terms of the likely surface pattern. This can be doneby assuming that the active portion of the microcarriers represents onlythe outer surface of the bead to a depth of about 20 angstroms. If it isalso assumed that the charged groups in the previously mentioned casesare evenly distributed throughout the beads, the previous ranges can beconverted to a charge capacity in this outer shell. Using this approach,the range of charge capacity found suitable is from about 0.012 meq/cm³to about 0.25 meq/cm³. This approach takes changes in microcarriervolume due to different charge densities into account.

Microcarriers having the required charge capacity can be prepared bytreating microcarriers formed from polymers containing pendant hydroxylgroups with an aqueous solution of an alkaline material and a tertiaryor quaternary amine. The beads can be initially swollen in an aqueousmedium without the other ingredients, or can be simply contacted with anaqueous medium containing the required base and amine. This method ofusing alkaline materials to catalyze the attachment of positivelycharged amino groups to hydroxyl-containing polymers is described inHartmann, U.S. Pat. No. 1,777,970.

Examples of suitable hydroxyl-containing polymers includepolysaccharides such as dextran, dextrin, starch, cellulose, polyglucoseand substituted derivatives of these. Certain synthetic polymers such aspolyvinyl alcohol and hydroxy-substituted acrylates or methacrylates,such as hydroxyethyl methacrylate, are also suitable. Dextran, andespecially crosslinked dextran in the form of small spheres or beads, isparticularly preferred because it is commercially available, relativelyinexpensive, and produces microcarriers which support excellent cellgrowth.

Any material which is alkaline can be used for the reaction. The alkalimetal hydroxides, such as sodium or potassium hydroxide, are, however,the preferred alkaline substances.

Either tertiary or quaternary amines are suitable sources of positivelycharged groups which can be appended onto the hydroxy-containingpolymers. Particularly preferred materials are chloro- orbromo-substituted tertiary amines or salts thereof, such asdiethylaminoethylchloride, diethylaminoethylbromide,dimethylaminoethylchloride, dimethylaminoethylbromide,diethylaminomethylchloride, diethylaminomethylbromide,di-(hydroxyethyl)-aminoethylchloride,di-(hydroxyethyl)-aminoethylbromide,di-(hydroxyethyl)-aminomethylchloride,di-(hydroxyethyl)-aminomethylbromide, β-morfolinoethylethylchloride,β-morfolinoethylbromide, β-morfolinomethylchloride,βmorfolinomethylbromide and salts thereof, for example, thehydrochlorides.

A wide range of reaction temperatures and times may be used. It ispreferred to carry out the reactions at temperatures of about between18° C. and 65° C. However, other temperatures can be used. The reactionkinetics depend to a large extent, of course, upon the reactiontemperature and the concentration of reactants. Both the time andtemperature do affect the final exchange capacity achieved.

The reason that the charge capacity of the microcarriers is so criticalin cell growth is not thoroughly understood. While not wishing to bebound by this theory, it is possible that the charge capacity at thesurface causes certain local discontinuities of medium composition whichare the major controlling influence in microcarrier culture cell growth.Nevertheless, this is not meant to rule out other possibilities.

There may be certain beads, of course, that will not be suitable forgood cell growth even though they have a charge capacity within one ofthe ranges specified. This may be due to side chains on the moietysupplying the charge capacity which are toxic or otherwise deleteriousfor cell growth, the presence of adsorbed or absorbed deleteriouscompositions or compounds, or it may be due to the porosity of the beador due to other reasons. If such beads are not suitable for cell growthexcept for the amount of charge capacity, the beads are not consideredto be "cell-growth microcarriers."

The invention is further illustrated by the following examples.

EXAMPLE 1 Preparation of Improved Microcarriers

Improved microcarriers can be produced as follows. Dry, uncharged,crosslinked dextran beads are seived to obtain those of approximately 75μm in diameter. One gram of this fraction is added to 10 ml of distilledwater and the beads are allowed to swell. An adequate commercial sourceof dry, crosslinked dextran is Sephadex G-50 from Pharmacia FineChemicals, Piscataway. N.J.

An aqueous solution containing 0.01 moles ofdiethylaminoethylchloride:chloride, twice recrystallized from methylenechloride, and 0.015 moles of sodium hydroxide is formed in a 10 mlvolume. This aqueous solution is then added to the swollen dextran beadsuspension, which is then agitated vigorously in a shaking water bathfor one hour at 60° C. After one hour, the beads are separated from thereaction mixture by filtration on Whatman filter paper No. 595 andwashed with 500 ml of distilled water.

Beads made by this procedure contain approximately 2.0 meq of chargecapacity per gram of dry, untreated cross-linked dextran. This chargecapacity can be characterized by measuring the anion exchange capabilityof the beads as follows. The bead preparations are washed thoroughlywith 0.1 normal HCl to saturate all exchange sites with Cl⁻ ions. Theyare then rinsed with 10⁻⁴ normal HCl to remove unbound chloride ions.Subsequently, the beads are washed with a 10% (w/w) sodium sulfatesolution to countersaturate the exchange sites with SO₄ =. The effluentof the sodium sulfate wash is collected and contains liberated chlorideions. This solution is titrated with 1 M silver nitrate using dilutepotassium chromate as an indicator.

After titration, the beads are washed thoroughly with distilled water,rinsed with the phosphate-buffered saline solution (PBS), suspended inPBS and autoclaved. This procedure yields hydrated beads ofapproximately 120-200 μm in diameter, which carry about 2.0 meq ofcharge capacity per gram of dry, untreated, crosslinked dextran.

EXAMPLE 2 Growth of Anchorage-Dependent Cells With Microcarriers of thisInvention Contrasted to Commercially Available Ion Exchange Resin

All cells were grown in Dulbecco's Modified Eagle's Medium. For growthof normal diploid fibroblasts, the medium was supplemented to 10% withfetal calf serum. For growth of primary and secondary chickenfibroblasts, the medium was supplemented with 1% chicken serum, 1% calfserum, and 2% tryptose phosphate broth (Difco Laboratories, Detroit,MI). Stocks were passaged on 100-mm plastic dishes (Falcon Plastics,Inc., Oxnard, CA).

Primary chicken embryo fibroblasts were prepared by mincing andsequentially trypsinizing 10-day embryos. Secondary chicken embryofibroblasts were prepared on the first day of primary confluence bytrypsinization. For cells grown in plastic dishes, doubling time wasabout 20 hours.

Diploid human fibroblasts derived from embryonic lung (HEL299, ATCC #CCL137) were obtained from the American Type Culture Collection, Rockville,MD. These cells had a doubling time of 19 hours in plastic dishes.

Microcarrier cultures were initiated simply by combining cells and beadsin stirred culture. 100-ml culture volumes in 250-ml glass spinnerbottles (6.5 cm in diameter) equipped with a 4.5-cm magnetically drivenTeflon® coated air bar (Wilbur Scientific, Inc., Boston, MA) were used.Stirring speed was approximately 90 rpm. Cultures were sampled directly,and samples were examined microscopically and photographed. Cells wereenumerated by counting nucleii using the modification of the method ofSanford et al. (Sanford, K. K., Earle, W. R., Evans, V. J., Waltz, H.K., and Shannon, J. E. (1951) J. Natl Cancer Inst. 11: 773.) asdescribed by van Wezel (van Wezel, A. L. (1973). Tissue Culture, Methodsand Applications. Kruse, P. F. and Patterson, M. R. (eds), pp. 372--377,Academic Press, New York).

Beads with attached cells were separated from the culture medium bypermitting the beads to settle at 1 g for a few minutes and thenaspirating the supernatant. This procedure greatly facilitated thereplacement of medium as well as facilitating the separation of cellsfrom microcarriers after trypsinization.

Commercial DEAE Sephadex A-50 was used as microcarrier for the diploidhuman fibroblasts and compared with carriers synthesized and titrated asdescribed in Example 1. For both bead types, carrier concentration was 2grams of dry, untreated, crosslinked dextran per liter. The chargecapacity of the DEAE Sephadex A-50 was 5.4 meq/g of dry, crosslinkeddextran, while that of the newly synthesized beads was 2.0 meq/g. Theresults are illustrated in FIG. 1.

For this cell type, loss of original inoculum on A-50 microcarriers wasmarked, while the fibroblasts attach, proliferate, and reach confluenceon the microcarriers of this invention in six days. This behavior agreeswell with the reported behavior of this cell type on standard plates. AsFIG. 1 shows, the final cell density achieved with the new microcarriersat 2 grams dry, crosslinked dextran/liter was 1.2×10⁶ cells/ml.

Cultures containing the new carriers demonstrated neither initial cellloss nor any inhibition in reaching confluence. More importantly, thecultures grew normally at higher microcarrier concentrations. In FIG. 2,for example, human fibroblasts and secondary chicken embryo fibroblastsare shown to reach saturation concentrations near 4×10⁶ cells/ml when 5grams of dry, crosslinked dextran per liter were used with the newcarriers having a charge capacity of 2.0 meq/g dextran. As can be seen,even at this relatively high microcarrier concentration, there was nosignificant loss of inoculum.

Secondary chick embryo fibroblasts were also grown at a microcarrierconcentration of 10 grams/liter. With the conditions described above, asaturation concentration of 6×10⁶ cells/ml was achieved; with additionto the medium of an additional 1% fetal calf serum, a saturationconcentration of 8×10⁶ cells/ml was achieved. There was no significantloss of cell inoculum.

Primary chick embryo fibroblasts were grown at a microcarrierconcentration of 5 and 10 grams/liter and the growth characteristicswere similar to those of the secondary chick fibroblasts, althoughslight inoculum losses were noted and somewhat longer lag times wereencountered.

Attempts were also made to grow secondary chick embryo fibroblasts underconditions similar to those used above except that DEAE-Sephadex A-50microcarriers at concentrations of 1 and 5 grams/liter were used. Nocell growth was detected and significant inoculum loss occurred.

EXAMPLE 3 Preparation of Microcarriers With Varying Amounts of Reactants

Batches of microcarriers were prepared by dissolvingdiethylaminoethylchloride:chloride and sodium hydroxide in 20 ml ofdistilled water. The solution was then poured over dry Sephadex G-50beads after which the beads were placed on a reciprocating shaker-watertable maintained at 60° C. One set of bead batches was treated with asolution containing 0.01 moles of the amine and 0.015 moles of sodiumhydroxide, whereas another set of batches was treated with a solutioncontaining 0.03 moles of the amine and 0.045 moles of sodium hydroxide.The reaction time was varied to produce different meq/g within eachbatch.

Diploid human fibroblasts (HEL299) were grown in suspension cultures ata microcarrier concentration of 5.0 grams dry, untreated crosslinkeddextran per liter following the procedures of Example 2 usingmicrocarriers having varying meq/gram selected from each batch.Subsequently, productivity (10⁶ cells grown/liter hour) was calculatedand plotted versus meq/gram for each batch of beads produced as above.Curves plotted using data obtained for both sets were similar in shape,having a general bell shape, but the curve from the batches treated withthe higher concentration of reactants had a somewhat sharper rise andfall. Carriers yielding excellent cell growth were produced from eachbatch.

EXAMPLE 4 Preparation of Microcarriers at Varying Amine/Alkali Ratios

This example illustrates further changes in the charge capacity whichcan be obtained by varying DEAE chloride:chloride/NaOH ratios. In thisexample, the procedures of Example 3 were followed except that a widerange of concentrations of sodium hydroxide was used while maintainingthe concentration of the diethylaminoethylchloride:chloride at 0.01moles per 20 ml. The concentrations used for the sodium hydroxide were0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.02, 0.03, 0.05, 0.75, 0.10moles per 20 ml.

A plot was made of meq/gram after 1.25 hours at 60° C. versusconcentration of sodium hydroxide. It was observed from the plot thatconcentrations of sodium hydroxide below about 0.01 produced nodetectable charge capacity. Charge capacity rose quickly, however, withincreases in concentration and reached a maximum of around 2.3 meq/gramdry, crosslinked dextran at a concentration of about 0.014 moles sodiumhydroxide. Charge capacity then declined in an almost linearrelationship to a value of about 1. meq/gram at a sodium hydroxideconcentration of about 0.10 moles. Thus, a change in reaction kineticstakes place when the ratio of DEAE Chloride: chloride to sodiumhydroxide is varied at a constant concentration of DEAEchloride:chloride and crosslinked dextran.

EXAMPLE 5 Human Interferon Production in Cells Grown on ImprovedMicrocarriers

The ability of microcarrier grown cells to produce human interferon isdescribed herein. Cells used for the production of human interferon werenormal diploid human foreskin fibroblasts, FS-4. These fibroblasts weregrown in microcarrier cultures using procedures as in Example 2.Microcarriers prepared and titrated according to Example 1 were used ata concentration of 5 grams of dry, crosslinked dextran/liter. The mediumused for culture growth was DMEM supplemented with 10% fetal calf serum.

In 8 to 10 days, cultures ceased growing. At this point, growth mediumwas removed. Cultures were washed 1-4 times with 100 ml of serum-freeDMEM. The cells were then ready for interferon induction. This wasaccomplished by adding to the cultures 50 ml of serum-free DMEM mediumcontaining 50 μg/ml cyclohexamide, and varying amounts of poly I poly Cinducer. After 4 hours, Actinomycin D was added to the cultures to afinal concentration of 1 μg/ml.

Five hours after the onset of induction, inducing medium was decantedand cultures were washed 3-4 times with 100 ml of warm serum-free DMEM.Cultures were replenished with 50 ml of DMEM containing 0.5% humanplasma protein. Cultures were incubated under standard conditions for anadditional 18 hours. At this time, cultures were decanted, and thedecanted medium was assayed for interferon activity. Interferon activitywas assayed by determining the 50% level of cell protection for samplesand standard solutions, for FS-4 fibroblasts challenged with VesicularStomatitis Virus (VSV), Indiana strain. The results of interferonproduction runs are presented in tabular form below.

    ______________________________________                                        Inducer      Cell Concentration                                               Concentration                                                                              During Production                                                                            Interferon                                        (μg/ml)   (cells/ml      (U/10.sup.6 cells)                                ______________________________________                                        4            2.0 × 10.sup.6                                                                         39                                                5            2.6 × 10.sup.6                                                                         378                                               25           2.6 × 10.sup.6                                                                         886                                               50           2.0 × 10.sup.6                                                                         ˜5000                                       ______________________________________                                    

These data are each from a separate run and are not intended todemonstrate any correlation to inducer concentration.

EXAMPLE 6 Growth of Cells on Improved Microcarriers for the Purpose ofProducing Viruses

The ability of microcarrier grown cells to produce a virus is describedhere. Primary and secondary chicken embryo fibroblasts were grown inmicrocarrier culture according to the procedure described in Example 2with the primary cells grown at 10 grams/liter and the secondary at 5grams/liter microcarrier concentration. To initiate virus production,growth medium was removed, and the cultures were washed twice with 100ml of serum free DMEM. Infection of cells with Sindbis virus took placein 50 ml of DMEM supplemented with 1% calf serum, 2% tryptose phosphatebroth, and enough Sindbis virus to equal an MOI (multiplicity ofinfection) of 0.05.

The virus was harvested 24 hours after infection, by collecting culturebroth, clarifying at low centrifugation, and freezing the supernatant.Virus production was assayed by plaque formation in a field of secondarychicken fibroblasts. The results of infecting these microcarriercultures were:

    ______________________________________                                                   All Concentration                                                             For Production                                                     Cell Type  (cells/ml)    (PFU/ml)   PFU/cell                                  ______________________________________                                        Secondary  4.0 × 10.sup.6                                                                         8.4 × 10.sup.9                                                                    2,100                                     Primary    1.4 × 10.sup.6                                                                        2.3 × 10.sup.10                                                                    16,000                                    Primary    6.0 × 10.sup.6                                                                        2.6 × 10.sup.10                                                                    5,000                                     ______________________________________                                    

Virus production was also established for the following virus/cell onmicrocarrier combinations: Polio/WI-38; Moloney MuLV/Cl-1 mouse andVSV/chick embryo fibroblasts.

EXAMPLE 7 Comparative Growth of Cells in Roller Bottles and withImproved Microcarriers for the Purpose of Producing Murine LeukemiaVirus Proviral DNA

The reverse-transcribed DNA of Moloney leukemia virus (M-MuLV) afterinfection of JLS-V9 cells, a mouse bone marrow line, was studied.

One technique involved growing cells in roller bottles. Cells were grownin roller bottle culture, the medium removed, and virus inoculumintroduced into the bottles. Shortly thereafter, the cultures were fedwith fresh medium, and 8-16 hours later extracted for eventualpurification of viral DNA. The cultures were washed with fresh bufferand the cell lysed with a solution containing the detergent sodiumdodecylsulfate. Subsequent cooling of the lysate and addition of salt toone molar caused co-precipitation of the detergent with high molecularweight DNA. The low molecular weight DNA remaining in the supernatantcould then be deproteinized and concentrated for further analysis.

A 50-roller bottle culture contained about 10⁹ cells. These wereinfected with about one-liter of viral inoculum titering at 3×10⁶plaque-forming units per ml. This resulted in a nominal multiplicity ofinfection of 1-3 and the infected cells yielded 5-20 nanograms ofvirus-specific DNA.

A simpler procedure was developed employing improved microcarriersaccording to this invention. A culture containing 10 grams of beads inone liter of growth medium was used. Upon reaching confluence, the 10⁹cells on the beads were infected by allowing the beads to settle out andreplacing the medium with 1 liter of virus inoculum. For extraction, thecells on the beads were washed with buffer and then placed in the SDScontaining buffer. After co-precipitation of the high molecular weightDNA with the detergent, the precipitate together with the beads werecentrifuged out and a supernatant extracted for further analysis. Theyield of viral DNA was comparable to that obtained in roller bottleculture and the labor involved was 5-10% of that required by rollerbottle culture.

EXAMPLE 8 Improved Microcarrier Production with DimethylaminoethylCharge Groups

A suitable microcarrier was produced by binding an alternate exchangemoiety to the dextran matrix utilized in Example 1. Dimethylaminoethylgroups (DMAE) were bound to a dextran matrix by the following procedure:1 gm of dextran beads (Pharmacia G-50), 50-75 μm in diameter, dry, wasadded to 10 ml of distilled water and the beads were allowed to swell.An aqueous solution containing 0.01 moles ofdimethylaminoethyl-chloride:chloride (Sigma Chemical Co.) and 0.015moles of sodium hydroxide was formed in a 10 ml volume. This aqueoussolution was added to the swollen dextran beads and this suspension wasthen agitated vigorously for one hour at 60° C. After reaction, the beadmass was titrated as in Example 1. This reaction binds 1.0 meq ofdimethylaminoethyl to the dextran mass. To produce microcarriers ofgreater degrees of substitution, the above reaction was carried out, andthe bead mass washed thoroughly with water. With excess water filteredoff, the bead mass was weighed so as to determine the amount of waterbeing retained by the bead mass. To this bead mass was added theappropriate amount of fresh reagents (i.e., DMAE-CL:CL, and NaOH) sothat the final concentration of DMAE, and NaOH in these succeedingreaction mixtures were identical to those initially used.

In this manner, a series of microcarriers were prepared at 1.0, 2.0, 2.5and 3.5 meq DMAE/gm unreacted dextran. Cells (HEL 299) were grown inmicrocarrier culture (5 gm/l) with these microcarriers according to theprocedures in Example 2. The results are tabulated in the followingtable:

    ______________________________________                                        Degree of                                                                     Substitution                                                                  (meq/gm)     Cell Spreading                                                                              Net Growth                                         ______________________________________                                        1.0          -             -                                                  2.0          -             -                                                  2.5          +             +                                                  3.2          +             -                                                  ______________________________________                                    

As expected, cell growth is related to the degree of substitution withcharge carrying groups. At too high a degree of substitution, no cellgrowth occurs, although attachment and spreading takes place. At too lowa degree of substitution, cell adhesion to the surface is not sufficientto allow proper spreading and growth.

EXAMPLE 9 Improved Microcarriers Having Positively Charged PhosphoniumGroups

Improved microcarriers were also prepared using non-amine exchangegroups as follows. One gram of dry dextran beads were prepared andswollen with water as in Example 1. To the swollen beads were added 5 mlof a saturated aqueous solution of triethyl-(ethyl-bromide)-phosphonium(TEP), ##STR1## and 5 ml of a 3 molar solution of sodium hydroxide. Thisslurry was reacted at 65° C. A series of microcarriers were prepared at1.1, 1.7 and 2.9 meq/gm. The microcarriers at 1.1 meq/gm were preparedby reaction at the above conditions for 4 minutes. The 1.7 meq/gmmicrocarrier was reacted for 1 hour, and the 2.9 meq/gm microcarrierswere reacted successively 3 times as described in Example 7. Amicrocarrier cell culture at 5 gm/liter was established for each ofthese carriers with a continuous cell type, JLS-V9 and compared to thiscell's growth on improved DEAE-microcarriers prepared as in Example 3.The results are tabulated in the following table.

    ______________________________________                                                     Cell Attachment                                                  meq/gram     and Spreading  Net Growth                                        ______________________________________                                        DEAE                                                                          0.9          +              +                                                 1.7          +              +                                                 3.8          +              -                                                 TEP                                                                           1.1          +              +                                                 1.7          +              +                                                 2.9          +              -                                                 ______________________________________                                    

It will be recognized by those skilled in the art that there are certainequivalents to the specific techniques, materials, etc., describedherein, and these are considered to be part of this invention and areintended to be covered by the following claims. Additionally, while mostof the description herein has been limited to the use of the improvedmicrocarriers for growth of anchorage-dependent cells, they can also beused, of course, for the growth of other cell types.

What is claimed is:
 1. Cell culture microcarriers having a degree ofsubstitution thereon with positively-charged chemical moietiessufficient to provide a charge capacity of from about 0.1 to about 4.5meq/gram of dry, untreated microcarriers.
 2. Cell culture microcarrierscomprising crosslinked dextran beads having a sufficient amount ofpositively charged groups thereon to provide a charge capacity ofbetween about 0.1 and about 4.5 meq/gram of dry, crosslinked dextranbeads.
 3. Cell culture microcarriers of claim 2 wherein said positivelycharged groups comprise diethylaminoethyl groups.
 4. Cell culturemicrocarriers comprising a reaction product of crosslinked dextran beadsand an aqueous solution of a tertiary or quaternary amine and a base,said aqueous solution having an amount and ratio of amine and basesufficient to provide said microcarriers with an exchange capacity offrom about 0.1 to about 4.5 meq/gram of dry dextran.
 5. Cell culturemicrocarriers of claim 4 wherein said amine comprises diethylaminoethyl.6. Cell culture microcarriers of claim 5 wherein said base comprisessodium hydroxide.
 7. A method of producing cell culture microcarrierscomprising soaking crosslinked dextran beads in an aqueous solution of atertiary or quaternary amine and a base until said beads are substitutedwith a sufficient amount of amine moieties to produce an exchangecapacity thereon of from about 0.1 to about 4.5 meq/gram of dry dextran.8. A method of claim 7 wherein said amine comprises diethylaminoethyl.9. A method of claim 8 wherein said base comprises sodium hydroxide.